WO2019224012A1 - Method and kit for predicting the outcome of an assisted reproductive technology procedure - Google Patents
Method and kit for predicting the outcome of an assisted reproductive technology procedure Download PDFInfo
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- WO2019224012A1 WO2019224012A1 PCT/EP2019/061967 EP2019061967W WO2019224012A1 WO 2019224012 A1 WO2019224012 A1 WO 2019224012A1 EP 2019061967 W EP2019061967 W EP 2019061967W WO 2019224012 A1 WO2019224012 A1 WO 2019224012A1
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- lactobacillus
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the invention relates to the field of human reproduction, more in particular to situations in which human reproduction is failing.
- the present invention provides a reliable and highly accurate method for predicting the chance that an assisted reproductive technology (ART) procedure, such as an in vitro fertilization and intra-cytoplasmic sperm injection (ICSI) procedure will not lead to a successful pregnancy. It also provides means and methods for predicting the chance that an assisted reproductive technology (ART) procedure, such as an in vitro fertilization and intra-cytoplasmic sperm injection (ICSI) procedure will lead to a successful pregnancy.
- ART assisted reproductive technology
- ICSI in vitro fertilization and intra-cytoplasmic sperm injection
- Sub-fertility affects 10 to 15% of couples in the western world. This sub-fertility can in half of the cases be attributed to causes related to the female reproductive system, in 20-26% to the male and in 25-30% the cause is unknown (Evers, J.L., 2002, Lancet 360:151 -159). Many couples turn to an assisted reproductive technology (ART) procedure such as in vitro fertilization (IVF) or intra-cytoplasmatic sperm injection (ICSI) to fulfil their child-wish.
- ART assisted reproductive technology
- IVF in vitro fertilization
- ICSI intra-cytoplasmatic sperm injection
- EP 2742359 B1 describes a method for predicting the chance of a successful or unsuccessful pregnancy in a subject, based on the relative amount of bacteria belonging to the group of lactobacillaceae and bacteria belonging to a species of Staphylococcus in a urine or vaginal sample.
- the invention relates to a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for the presence of Gardnerella vaginalis 1ST 1 and additionally for at least one of the following parameters:
- the invention also relates to a kit for performing a method according to the invention comprising a forward primer CTGGATCACCTCCTTTCTAWG (SEQ ID NO:
- Gardnerella vaginalis 1ST 1 DNA amplification product has a length of 428 - 430 nucleotides, and wherein W denotes an A or a T and wherein R denotes an A or a G.
- ART assisted reproductive technology
- the subject has a high likelihood of not becoming pregnant as a result of the ART procedure if the sample comprises Gardnerella vaginalis 1ST 1 and at least one of the following applies:
- the values chosen between 15% and 25%, 25% and 45% and 18% and 38% respectively are also often referred to as a cut-off values or a threshold values. It may be chosen such that the method provides the desired specificity and sensitivity. A skilled person is well aware of the meets and bounds of determining a suitable value.
- the presence of Gardnerella vaginalis is determined as well as the relative abundance of Lactobacillus species, the relative abundance of Lactobacillus jensenii and the relative abundance of Proteobacteria.
- the invention relates to a method as described above, wherein the Gardnerella vaginalis is Gardnerella vaginalis 1ST 1.
- Gardnerella vaginalis IST1 is defined herein as a specific Gardnerella species that may be identified by performing vaginal microbial population analysis using amplification of the intergenic spaces (IS), according to the protocol provided by the manufacturer (IS-pro technique, IS-Diagnostics, Amsterdam, the Netherlands).
- IS-pro is an eubacterial technique based on the detection and categorisation of the length of the 16S-23S rRNA gene IS region. The length of this IS region is specific for each microbial species.
- Gardnerella vaginalis IST1 is hereby further defined as a species of Gardnerella vaginalis that results in a specific IS-fragment with a length of 428, 429 or 430 nucleotides when primers according to SEQ ID NO: 1 and SEQ ID NO: 2 are used.
- ART procedure is used herein to indicate an artificial reproductive technology.
- the term relates to in Vitro Fertilization (IVF), Intra Cytoplasmic Sperm Injection (ICSI) and Intra Uterine Insemination (IUI).
- relative abundance is used to indicate a fraction of the total amount or number of bacteria in a sample.
- the fraction is either expressed as a percentage (%) or as a number between 0 and 1.
- the term“high likelihood” in respect of predicting the chance of the success or failure of an ART procedure is used herein to indicate that the predicted success or failure rate is higher than in the general population of women undergoing an ART procedure.
- the likelihood of not becoming pregnant is referred to as “increased” if the subject has a higher than 60% chance, such as 65% chance of not becoming pregnant as a result of the ART procedure if the criteria for a negative prediction as described herein are fulfilled. Higher than 65% in this respect includes for instance higher than 77%, such as 88% or higher or even 94% or higher.
- the likelihood of becoming pregnant is increased if the subject has a higher than 35% chance of becoming pregnant as a result of the ART procedure if the criteria for a positive prediction as described herein are fulfilled.
- Higher than 35% in this respect means 41%, 49% or even 50% or more.
- Gardnerella is a genus of Gram-variable-staining facultative anaerobic bacteria of which Gardnerella vaginalis is the only species.
- the organisms are small (1.0- 1.5 mm in diameter) nonspore-forming, nonmotile coccobacilli. Once classified as
- G. vaginalis grows as small, circular, convex, gray colonies on chocolate agar; it also grows on HBT agar.
- a selective medium for G. vaginalis is colistin-oxolinic acid blood agar. Determining the presence of Gardnerella vaginalis is preferably done by PCR, such as quantitative PCR.
- Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria. They are a major part of the lactic acid bacteria group (i.e. they convert sugars to lactic acid). In humans, they constitute a significant component of the microbiota at a number of body sites, such as the digestive system, urinary system, and genital system. In women of European ancestry, Lactobacillus species are normally a major part of the vaginal microbiota. Lactobacillus forms biofilms in the vaginal and gut microbiota, allowing them to persist during harsh environmental conditions and maintain ample populations. Lactobacillus exhibits a mutualistic relationship with the human body as it protects the host against potential invasions by pathogens, and in turn, the host provides a source of nutrients.
- Lactobacillus species is used herein to refer to all Lactobacillus species collectively.
- Lactobacillus jensenii is a common inhabitant of the lower reproductive tract in healthy women. In a normal population, L. jensenii makes up to about 23% of vaginal microflora that is naturally occurring.
- the Proteobacteria are a major phylum of bacteria. They are gram-negative bacteria. This means they do not retain the violet dye in the Gram staining protocol. In a Gram stain test, a counterstain (commonly safranin) is added after the crystal violet, colouring all gram-negative bacteria with a pink colour. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their cell walls.
- Proteobacteria include a wide variety of pathogens, such as Escherichia coli, Salmonella, Vibrio, Helicobacter, and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation. The group is defined primarily in terms of ribosomal RNA (rRNA) sequences.
- rRNA ribosomal RNA
- the relative abundance of a particular species or genus of bacteria has to be compared with a predetermined reference value or cut-off value.
- the predetermined reference value may be any suitable cut-off value. This process of determining a suitable cut-off value is well within the skills of a skilled person and can easily be determined empirically by the skilled person.
- it is a value derived from the bacterial composition of samples obtained from a comparable population as the test population. Even more preferred is a reference value obtained from an average value of several independent experiments of ART procedures in a reference population. The skilled person is aware of the particulars of determining reference values for measuring and determining the relative abundance of bacteria.
- the predetermined reference value may be empirically determined or arbitrarily chosen in order to achieve appropriate specificity and/or sensitivity of the method.
- a skilled person is fully aware how to choose an appropriate reference value.
- a skilled person will know how to alter the predetermined reference value in order to obtain the desired specificity and sensitivity of the method.
- the first predetermined reference value may be between 15 and 25%, such as 20%
- the second predetermined reference value may be between 25 and 45%, such as 35%
- the third predetermined reference value may be between 18 and 38%, such as 28%.
- a method according to the invention as disclosed above produces highly reliable results, i.e. it failed to predict an unsuccessful outcome of the ART procedure in only 2 cases. In these two cases, the ART procedure resulted in a pregnancy which is considered the desired outcome; i.e. a successful outcome.
- the total average costs for an ART procedure such as IVF or ICSI are in the order of €5.000.
- applying the method according to the invention would have saved on average €900 per IVF/ICSI patient, or in other terms, the costs of the procedure would have been reduced with 18%.
- the invention also relates to a method as described above wherein the presence of Gardnerella vaginalis, preferably G. vaginalis 1ST, such as G. vaginalis 1ST 1 is determined in the sample and wherein the subject has a high likelihood of not becoming pregnant as a result of the ART procedure if the sample comprises Gardnerella vaginalis, preferably G. vaginalis 1ST 1.
- the accuracy and other features of this method as described above could even be further improved by applying a method wherein at least one of the following parameters is measured:
- the subject has a high likelihood of not becoming pregnant as a result of the ART procedure if the sample comprises Gardnerella vaginalis IST 1 and at least one of the following applies:
- criterion 1 was combined with the criterion of the relative abundance of Proteobacteria (criterion 4) being above 18%, then an extra 5 samples of these 32 could be correctly predicted. Other combinations of these criteria also yielded an improvement in the method.
- Table 7 provides the numbers of correct predictions depending on the criteria used. If all 4 criteria were used, all 32 samples were detected.
- Table 7 Correct prediction of not becoming pregnant based on different criteria.
- Lactobacillus crispatus is a common inhabitant of the lower reproductive tract in healthy women. In a normal population, L. crispatus is the dominant species in more than 30% of all women of reproductive age.
- Lactobacillus iners is also a species in the genus Lactobacillus. It is a Gram positive, catalase-negative, facultatively anaerobic rod-shaped bacterium. Lactobacillus iners is a normal inhabitant of the lower reproductive tract in healthy women. The genomes of at least 15 strains have been sequenced and encode between 1 ,152 and 1 ,506 proteins. Therewith this species has one of the smallest Lactobacillus genomes compared to other species, such as L. crispatus, which typically encodes more than twice as many proteins.
- a subject had a high likelihood of not becoming pregnant as a result of an ART procedure, if the relative abundance of Lactobacillus crispatus was above a fourth predetermined reference value.
- This fourth predetermined reference value was preferably chosen between 50 and 70%, such as 60%. When 60% was taken as the fourth reference value, 65 women fulfilled this criterion, of which 15 became pregnant as a result of the ART procedure (Table 6). This is a failure rate of 77%, which is higher than the failure rate in the entire group (Table 5). It was also found that a subject had a high likelihood of becoming pregnant as a result of the ART procedure, if the relative abundance of Lactobacillus crispatus was below a fifth predetermined reference value.
- This fifth predetermined reference value was preferably chosen between 50 and 70%, such as 60%.
- 60% was taken as the fifth reference value, 127 women fulfilled this criterion, of which 52 became pregnant as a result of the ART procedure (Table 6). This is a success rate of 41 %, which is higher than the success rate in the entire group (Table 5).
- This sixth predetermined reference value was preferably chosen between 50 and 70%, such as 60%.
- 60% was taken as the sixth reference value, 38 women fulfilled this criterion, of which 19 became pregnant as a result of the ART procedure (Table 5). This is a success rate of 50%, which is higher than the success rate in the entire group (Table 5).
- ART assisted reproductive technology
- ART assisted reproductive technology
- ART assisted reproductive technology
- a is a value between -0.55 and -0.70
- b is a value between 0.80 and 0.90
- c is a value between -0.50 and -0.65
- d is a value between 0.3 and 0.45.
- 77 of the 192 women from the study described herein were found to fulfill the criterion, of which 38 (49%) became pregnant as a result of the ART procedure (Table 5).
- Figure 1 Scatter plot of data obtained with one of the methods exemplified herein wherein the likelihood of a pregnancy is increased if [LC] is below 60% and wherein
- Example 1 Study population
- vaginal swab by themselves prior to the start of the IVF or IVF-ICSI procedure.
- a self-collecting method was chosen, because it is minimal invasive for the patient and therefore suitable for use in the daily practice.
- the vaginal samples were taken with FLOQSwabsTM (Copan Italia S.p.A., Italy) and the participants were instructed to insert the swab 3-5 centimetre into the vagina, then to rub the swab along the vaginal wall for 10-15 seconds. After this procedure the swabs were immediately placed in Eppendorf tubes filled with reduced transport fluid (RTF) buffer, obtained from IS-Diagnostics (IS-Diagnostics, Amsterdam, the Netherlands). Up to the analysis, the samples were stored at -20 to -80 oC degrees in the freezer.
- RTF reduced transport fluid
- Urine samples were collected in a sterile urine collecting device of 100 ml. A 10 ml sample was centrifuged for 10 minutes at 1500 RCF. The supernatant was decanted and the pellet was re-suspended in 3 ml urine. The re-suspended sample was stored for further processing at -20 degrees Celsius.
- DNA extraction was performed from the vaginal swabs with the Chemagen (Chemagen, Baesweiler, Germany) automated DNA extraction machine using the buccal swab extraction kit according to the manufacturer’s instructions. First the swabs were thawed and vortexed. 200 mI of sample was incubated with 200 mI Chemagen lysisbuffer and 10 mI Proteinase K (Qiagen, Hilden, Germany) at 56 degrees Celsius while shaking at 500 rpm. DNA was extracted using the protocol buccal Swab Prefilling. Elution of DNA was in 100mI of Chemagen Elution buffer.
- DNA was extracted from concentrated urine suspensions with the Chemagen (Perkin-Elmer, Baesweiler, Germany) automated DNA extraction machine using the buccal swab extraction kit according to the manufacturer’s instructions.
- Chemagen Perkin-Elmer, Baesweiler, Germany
- urine samples were thawed and vortexed.
- 200 mI of sample was incubated with 200 mI Chemagen lysis buffer and 10 mI Proteinase K at 56 degrees Celsius while shaking at 500 rpm.
- Elution of DNA was in 100mI of Chemagen Elution buffer.
- IS-pro is an eubacterial technique based on the detection and
- the length of the 16S-23S rRNA gene IS region is specific for each microbial species.
- Phylum-specific fluorescently labelled PCR primers are used for taxonomic classification.
- the procedure consists of two separate standard PCRs: the first PCR mixture contains two different fluorescently labelled forward primers targeting different bacterial groups and three reverse primers providing universal coverage for those groups.
- the first forward primer is specific for the phyla Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV), and the second labeled forward primer is specific for the phylum Bacteroidetes.
- a separate PCR with a labeled forward primer combined with seven reverse primers is specific for the phylum Proteobacteria [Budding, E. et al., J. Clin. Microbiol. (2016) 54: 934-943]
- Pregnancy outcome after the first embryo transfer was used as endpoint. Ongoing pregnancy was defined as a fetus with heart activity established with the use of an ultrasound between 7-9 weeks of gestation.
- Gardnerella vaginalis IST1 was identified by performing vaginal microbial population analysis with the IS-pro technique. G.vaginalis IST1 was detected by presence of a specific IS-fragment with a length of 428-430 nucleotides.
- Table 4 Microbial composition of vaginal flora; prediction of chance of failure of ART procedure.
- a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for at least one of the following parameters:
- the sample comprises Gardnerella vaginalis or
- Proteobacteria is above a third predetermined reference value.
- the first predetermined reference value is 20%
- the second predetermined reference value is 35%
- the third predetermined reference value is 28%
- Lactobacillus crispatus [LC] and Lactobacillus iners [LI] are determined and expressed as fractions between 0 and 1 , and wherein the likelihood of becoming pregnant is increased if:
- a is a value between -0.55 and - 0.70
- b is a value between 0.8 and 0.9
- c is a value between -0.50 and -0.65
- d is a value between 0.30 and 0.45.
- Kit for performing a method as described herein comprising a forward primer
- sample is a vaginal swab and/or wherein the mammalian subject is human.
- ART procedure is an in vitro fertilization (IVF) procedure, such as an intra-cytoplasmic sperm injection (ICSI) procedure.
- IVF in vitro fertilization
- ICSI intra-cytoplasmic sperm injection
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CN201980043008.7A CN112384633A (en) | 2018-05-22 | 2019-05-09 | Methods and kits for predicting assisted reproductive technology procedure outcome |
AU2019273443A AU2019273443A1 (en) | 2018-05-22 | 2019-05-09 | Method and kit for predicting the outcome of an assisted reproductive technology procedure |
US17/057,033 US20210324451A1 (en) | 2018-05-22 | 2019-05-09 | Method and kit for predicting the outcome of an assisted reproductive technology procedure |
EP19725656.3A EP3797174A1 (en) | 2018-05-22 | 2019-05-09 | Method and kit for predicting the outcome of an assisted reproductive technology procedure |
CA3099513A CA3099513A1 (en) | 2018-05-22 | 2019-05-09 | Method and kit for predicting the outcome of an assisted reproductive technology procedure |
EA202092443A EA202092443A1 (en) | 2018-05-22 | 2019-05-09 | METHOD AND KIT FOR FORECASTING THE RESULT OF AUXILIARY REPRODUCTIVE TECHNOLOGIES |
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